Structured Review

TaKaRa pegfp c3
hZNF-134 over expression enhanced the transcriptional potential of LTR and increased viral titers. (A) Luciferase activity was measured in the presence or absence of Tat following transfection with <t>pEGFP-C3</t> or ZNF-134-GFP-C3 vector into HEK293T cells along with the reporter construct, pLTR-luc. Luciferase activity was measured in terms of relative luminescence units (RLU) from HEK293T cell lysates after 48 hours and plotted as fold increase in the luciferase activity. Cells over-expressing hZNF-134-GFP showed increased luciferase activity when compared to GFP alone. (B) pNL4-3 was transfected in HEK293T cells with either GFP or hZNF-134-GFP. After 48 hours, the viral transcription was measured by qRT-PCR. The plot shows that viral transcripts increased in the presence of over-expressed hZNF-134. (C) HEK293T cells over-expressing either GFP or hZNF-134-GFP were transfected with pNL4-3 vector and after 48 hours scored for viral p24 by ELISA. Inset shows the Western blot confirming the over-expression of either GFP or hZNF-134-GFP in HEK293T cells. For loading control, expression of GAPDH was detected by Western blots in both the cases. Over-expression of hZNF-134 increased viral titers. All the transfection efficiencies were normalized to pRL-TK transfections. All experiments were done more than three times and error bars represents mean ± SD. Student's t -test was performed and *p
Pegfp C3, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pegfp c3/product/TaKaRa
Average 91 stars, based on 19 article reviews
Price from $9.99 to $1999.99
pegfp c3 - by Bioz Stars, 2020-05
91/100 stars

Images

1) Product Images from "Mycobacterial and HIV Infections Up-Regulated Human Zinc Finger Protein 134, a Novel Positive Regulator of HIV-1 LTR Activity and Viral Propagation"

Article Title: Mycobacterial and HIV Infections Up-Regulated Human Zinc Finger Protein 134, a Novel Positive Regulator of HIV-1 LTR Activity and Viral Propagation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0104908

hZNF-134 over expression enhanced the transcriptional potential of LTR and increased viral titers. (A) Luciferase activity was measured in the presence or absence of Tat following transfection with pEGFP-C3 or ZNF-134-GFP-C3 vector into HEK293T cells along with the reporter construct, pLTR-luc. Luciferase activity was measured in terms of relative luminescence units (RLU) from HEK293T cell lysates after 48 hours and plotted as fold increase in the luciferase activity. Cells over-expressing hZNF-134-GFP showed increased luciferase activity when compared to GFP alone. (B) pNL4-3 was transfected in HEK293T cells with either GFP or hZNF-134-GFP. After 48 hours, the viral transcription was measured by qRT-PCR. The plot shows that viral transcripts increased in the presence of over-expressed hZNF-134. (C) HEK293T cells over-expressing either GFP or hZNF-134-GFP were transfected with pNL4-3 vector and after 48 hours scored for viral p24 by ELISA. Inset shows the Western blot confirming the over-expression of either GFP or hZNF-134-GFP in HEK293T cells. For loading control, expression of GAPDH was detected by Western blots in both the cases. Over-expression of hZNF-134 increased viral titers. All the transfection efficiencies were normalized to pRL-TK transfections. All experiments were done more than three times and error bars represents mean ± SD. Student's t -test was performed and *p
Figure Legend Snippet: hZNF-134 over expression enhanced the transcriptional potential of LTR and increased viral titers. (A) Luciferase activity was measured in the presence or absence of Tat following transfection with pEGFP-C3 or ZNF-134-GFP-C3 vector into HEK293T cells along with the reporter construct, pLTR-luc. Luciferase activity was measured in terms of relative luminescence units (RLU) from HEK293T cell lysates after 48 hours and plotted as fold increase in the luciferase activity. Cells over-expressing hZNF-134-GFP showed increased luciferase activity when compared to GFP alone. (B) pNL4-3 was transfected in HEK293T cells with either GFP or hZNF-134-GFP. After 48 hours, the viral transcription was measured by qRT-PCR. The plot shows that viral transcripts increased in the presence of over-expressed hZNF-134. (C) HEK293T cells over-expressing either GFP or hZNF-134-GFP were transfected with pNL4-3 vector and after 48 hours scored for viral p24 by ELISA. Inset shows the Western blot confirming the over-expression of either GFP or hZNF-134-GFP in HEK293T cells. For loading control, expression of GAPDH was detected by Western blots in both the cases. Over-expression of hZNF-134 increased viral titers. All the transfection efficiencies were normalized to pRL-TK transfections. All experiments were done more than three times and error bars represents mean ± SD. Student's t -test was performed and *p

Techniques Used: Over Expression, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Construct, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot

hZNF-134-GFP is localized in the nucleus of HEK293T and Astrocytoma 1321N1 cells. HEK293T and 1321N1 cells were transfected with expression plasmids pEGFP-C3 or ZNF-134-GFP-C3. The cells were fixed after 48 hours and visualized under confocal microscopy. Panels show Trans, DAPI, GFP, and Merged images. hZNF-134-GFP protein was localized in the nucleus of HEK293T and 1321N1 cells whereas GFP control showed both nuclear and cytoplasmic localization. Localization was confirmed by 3 independent experiments.
Figure Legend Snippet: hZNF-134-GFP is localized in the nucleus of HEK293T and Astrocytoma 1321N1 cells. HEK293T and 1321N1 cells were transfected with expression plasmids pEGFP-C3 or ZNF-134-GFP-C3. The cells were fixed after 48 hours and visualized under confocal microscopy. Panels show Trans, DAPI, GFP, and Merged images. hZNF-134-GFP protein was localized in the nucleus of HEK293T and 1321N1 cells whereas GFP control showed both nuclear and cytoplasmic localization. Localization was confirmed by 3 independent experiments.

Techniques Used: Transfection, Expressing, Confocal Microscopy

2) Product Images from "Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice"

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

Journal: Endocrinology

doi: 10.1210/en.2015-1556

Determination of the control strength of ADAM17 on ACE2 activity. 832/13 cells were cotransfected with 25 ng/well mACE2/pcDNA3.1 and 2 μg/well of a mix of pAd17 and pAd17E406A. The pAd17 content in the mix were 0, 0.4, 0.8, 1.2, 1.6, and 2 μg/well in four experiments and 0, 0.05, 0.1, 0.2, 0,4, and 2 μg/well in eight experiments. ADAM17 activities (A) and ACE2 activities (B) were measured. C, For estimation of transfection efficiencies, green fluorescent 832/13 cells transfected with 2 μg pEGFP-C3 and 25 ng mACE/pcDNA3.1 were determined as in the upper right panel compared with control cells in the upper left panel. To estimate cotransfection efficiency, cells cotransfected with a plasmid encoding the red fluorescent tdTomato and pAd17 (lower left panel) were compared to cells cotransfected with plasmids for eGFP and tdTomato expression (lower right panel). For the different amounts of pAd17 used in the transfection experiments, shed ACE2 (D) and cellular ACE2 (E) were plotted against the estimated ADAM17 activity of the transfected cells, ie, corrected for the transfection efficiency. Curves describing saturation kinetics were fitted to the data. F, Based on the model fits, the control strengths were calculated and plotted as a function of the ADAM17 activity of transfected cells.
Figure Legend Snippet: Determination of the control strength of ADAM17 on ACE2 activity. 832/13 cells were cotransfected with 25 ng/well mACE2/pcDNA3.1 and 2 μg/well of a mix of pAd17 and pAd17E406A. The pAd17 content in the mix were 0, 0.4, 0.8, 1.2, 1.6, and 2 μg/well in four experiments and 0, 0.05, 0.1, 0.2, 0,4, and 2 μg/well in eight experiments. ADAM17 activities (A) and ACE2 activities (B) were measured. C, For estimation of transfection efficiencies, green fluorescent 832/13 cells transfected with 2 μg pEGFP-C3 and 25 ng mACE/pcDNA3.1 were determined as in the upper right panel compared with control cells in the upper left panel. To estimate cotransfection efficiency, cells cotransfected with a plasmid encoding the red fluorescent tdTomato and pAd17 (lower left panel) were compared to cells cotransfected with plasmids for eGFP and tdTomato expression (lower right panel). For the different amounts of pAd17 used in the transfection experiments, shed ACE2 (D) and cellular ACE2 (E) were plotted against the estimated ADAM17 activity of the transfected cells, ie, corrected for the transfection efficiency. Curves describing saturation kinetics were fitted to the data. F, Based on the model fits, the control strengths were calculated and plotted as a function of the ADAM17 activity of transfected cells.

Techniques Used: Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Expressing

3) Product Images from "Intramembrane Proteolysis and Endoplasmic Reticulum Retention of Hepatitis C Virus Core Protein"

Article Title: Intramembrane Proteolysis and Endoplasmic Reticulum Retention of Hepatitis C Virus Core Protein

Journal: Journal of Virology

doi: 10.1128/JVI.78.12.6370-6380.2004

Expression plasmids used in this study. The genes encoding HCV proteins and their mutants were cloned into pcDNA3.1FlagHA, pcDNA3.1/ myc -His C, or pEGFP-C3 as described in Materials and Methods. Other plasmids are described in the text or in the other figure legends.
Figure Legend Snippet: Expression plasmids used in this study. The genes encoding HCV proteins and their mutants were cloned into pcDNA3.1FlagHA, pcDNA3.1/ myc -His C, or pEGFP-C3 as described in Materials and Methods. Other plasmids are described in the text or in the other figure legends.

Techniques Used: Expressing, Clone Assay

4) Product Images from "Mycobacterial and HIV Infections Up-Regulated Human Zinc Finger Protein 134, a Novel Positive Regulator of HIV-1 LTR Activity and Viral Propagation"

Article Title: Mycobacterial and HIV Infections Up-Regulated Human Zinc Finger Protein 134, a Novel Positive Regulator of HIV-1 LTR Activity and Viral Propagation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0104908

hZNF-134 over expression enhanced the transcriptional potential of LTR and increased viral titers. (A) Luciferase activity was measured in the presence or absence of Tat following transfection with pEGFP-C3 or ZNF-134-GFP-C3 vector into HEK293T cells along with the reporter construct, pLTR-luc. Luciferase activity was measured in terms of relative luminescence units (RLU) from HEK293T cell lysates after 48 hours and plotted as fold increase in the luciferase activity. Cells over-expressing hZNF-134-GFP showed increased luciferase activity when compared to GFP alone. (B) pNL4-3 was transfected in HEK293T cells with either GFP or hZNF-134-GFP. After 48 hours, the viral transcription was measured by qRT-PCR. The plot shows that viral transcripts increased in the presence of over-expressed hZNF-134. (C) HEK293T cells over-expressing either GFP or hZNF-134-GFP were transfected with pNL4-3 vector and after 48 hours scored for viral p24 by ELISA. Inset shows the Western blot confirming the over-expression of either GFP or hZNF-134-GFP in HEK293T cells. For loading control, expression of GAPDH was detected by Western blots in both the cases. Over-expression of hZNF-134 increased viral titers. All the transfection efficiencies were normalized to pRL-TK transfections. All experiments were done more than three times and error bars represents mean ± SD. Student's t -test was performed and *p
Figure Legend Snippet: hZNF-134 over expression enhanced the transcriptional potential of LTR and increased viral titers. (A) Luciferase activity was measured in the presence or absence of Tat following transfection with pEGFP-C3 or ZNF-134-GFP-C3 vector into HEK293T cells along with the reporter construct, pLTR-luc. Luciferase activity was measured in terms of relative luminescence units (RLU) from HEK293T cell lysates after 48 hours and plotted as fold increase in the luciferase activity. Cells over-expressing hZNF-134-GFP showed increased luciferase activity when compared to GFP alone. (B) pNL4-3 was transfected in HEK293T cells with either GFP or hZNF-134-GFP. After 48 hours, the viral transcription was measured by qRT-PCR. The plot shows that viral transcripts increased in the presence of over-expressed hZNF-134. (C) HEK293T cells over-expressing either GFP or hZNF-134-GFP were transfected with pNL4-3 vector and after 48 hours scored for viral p24 by ELISA. Inset shows the Western blot confirming the over-expression of either GFP or hZNF-134-GFP in HEK293T cells. For loading control, expression of GAPDH was detected by Western blots in both the cases. Over-expression of hZNF-134 increased viral titers. All the transfection efficiencies were normalized to pRL-TK transfections. All experiments were done more than three times and error bars represents mean ± SD. Student's t -test was performed and *p

Techniques Used: Over Expression, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Construct, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot

hZNF-134-GFP is localized in the nucleus of HEK293T and Astrocytoma 1321N1 cells. HEK293T and 1321N1 cells were transfected with expression plasmids pEGFP-C3 or ZNF-134-GFP-C3. The cells were fixed after 48 hours and visualized under confocal microscopy. Panels show Trans, DAPI, GFP, and Merged images. hZNF-134-GFP protein was localized in the nucleus of HEK293T and 1321N1 cells whereas GFP control showed both nuclear and cytoplasmic localization. Localization was confirmed by 3 independent experiments.
Figure Legend Snippet: hZNF-134-GFP is localized in the nucleus of HEK293T and Astrocytoma 1321N1 cells. HEK293T and 1321N1 cells were transfected with expression plasmids pEGFP-C3 or ZNF-134-GFP-C3. The cells were fixed after 48 hours and visualized under confocal microscopy. Panels show Trans, DAPI, GFP, and Merged images. hZNF-134-GFP protein was localized in the nucleus of HEK293T and 1321N1 cells whereas GFP control showed both nuclear and cytoplasmic localization. Localization was confirmed by 3 independent experiments.

Techniques Used: Transfection, Expressing, Confocal Microscopy

5) Product Images from "Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice"

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

Journal: Endocrinology

doi: 10.1210/en.2015-1556

Determination of the control strength of ADAM17 on ACE2 activity. 832/13 cells were cotransfected with 25 ng/well mACE2/pcDNA3.1 and 2 μg/well of a mix of pAd17 and pAd17E406A. The pAd17 content in the mix were 0, 0.4, 0.8, 1.2, 1.6, and 2 μg/well in four experiments and 0, 0.05, 0.1, 0.2, 0,4, and 2 μg/well in eight experiments. ADAM17 activities (A) and ACE2 activities (B) were measured. C, For estimation of transfection efficiencies, green fluorescent 832/13 cells transfected with 2 μg pEGFP-C3 and 25 ng mACE/pcDNA3.1 were determined as in the upper right panel compared with control cells in the upper left panel. To estimate cotransfection efficiency, cells cotransfected with a plasmid encoding the red fluorescent tdTomato and pAd17 (lower left panel) were compared to cells cotransfected with plasmids for eGFP and tdTomato expression (lower right panel). For the different amounts of pAd17 used in the transfection experiments, shed ACE2 (D) and cellular ACE2 (E) were plotted against the estimated ADAM17 activity of the transfected cells, ie, corrected for the transfection efficiency. Curves describing saturation kinetics were fitted to the data. F, Based on the model fits, the control strengths were calculated and plotted as a function of the ADAM17 activity of transfected cells.
Figure Legend Snippet: Determination of the control strength of ADAM17 on ACE2 activity. 832/13 cells were cotransfected with 25 ng/well mACE2/pcDNA3.1 and 2 μg/well of a mix of pAd17 and pAd17E406A. The pAd17 content in the mix were 0, 0.4, 0.8, 1.2, 1.6, and 2 μg/well in four experiments and 0, 0.05, 0.1, 0.2, 0,4, and 2 μg/well in eight experiments. ADAM17 activities (A) and ACE2 activities (B) were measured. C, For estimation of transfection efficiencies, green fluorescent 832/13 cells transfected with 2 μg pEGFP-C3 and 25 ng mACE/pcDNA3.1 were determined as in the upper right panel compared with control cells in the upper left panel. To estimate cotransfection efficiency, cells cotransfected with a plasmid encoding the red fluorescent tdTomato and pAd17 (lower left panel) were compared to cells cotransfected with plasmids for eGFP and tdTomato expression (lower right panel). For the different amounts of pAd17 used in the transfection experiments, shed ACE2 (D) and cellular ACE2 (E) were plotted against the estimated ADAM17 activity of the transfected cells, ie, corrected for the transfection efficiency. Curves describing saturation kinetics were fitted to the data. F, Based on the model fits, the control strengths were calculated and plotted as a function of the ADAM17 activity of transfected cells.

Techniques Used: Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Expressing

6) Product Images from "Novel restriction factor RNA-associated early-stage anti-viral factor (REAF) inhibits human and simian immunodeficiency viruses"

Article Title: Novel restriction factor RNA-associated early-stage anti-viral factor (REAF) inhibits human and simian immunodeficiency viruses

Journal: Retrovirology

doi: 10.1186/1742-4690-11-3

Over-expression of REAF confers restriction which is viral route of entry dependent. (A) Over-expression of REAF restricts early HIV-1 DNA production. HEK 293 T cells were transiently transfected with pEGFP-C3 or REAF-EGFP and the Western blot probed with α-GFP antibody. Transfection of REAF-EGFP results in expression of REAF (110, 160 and 250 kDa including EGFP tag (30 kDa). Endogenous REAF was knocked out of HeLa-CD4 cells using siRNA targeting its 3’ UTR. (B) Infected cells were measured following transient over-expression of REAF-EGFP or pEGFP-C3 and challenge with HIV-1 89.6 . Results are shown as FFU/ml and are mean ± s.d. of a representative experiment performed in duplicate. (C) Transient over-expression of REAF-EGFP in HeLa-CD4 cells results in decreased RU5 and late RT products 8 hr post challenge with HIV-1 89.6 . HIV-1 DNA copies are normalised to genomic GAPDH and presented per million cells. Results are mean ± s.d. of a representative experiment performed in duplicate. (D) Restriction by REAF is dependent upon viral route of entry. HIV-1 89.6Δenv was pseudotyped with VSV-G envelope and used to challenge HeLa-CD4 cells following REAF siRNA knockdown. p24 immunostaining was used to detect FFU compared with HIV-1 89.6 . Results are shown as FFU/ml and are mean ± s.d. of a representative experiment performed in duplicate.
Figure Legend Snippet: Over-expression of REAF confers restriction which is viral route of entry dependent. (A) Over-expression of REAF restricts early HIV-1 DNA production. HEK 293 T cells were transiently transfected with pEGFP-C3 or REAF-EGFP and the Western blot probed with α-GFP antibody. Transfection of REAF-EGFP results in expression of REAF (110, 160 and 250 kDa including EGFP tag (30 kDa). Endogenous REAF was knocked out of HeLa-CD4 cells using siRNA targeting its 3’ UTR. (B) Infected cells were measured following transient over-expression of REAF-EGFP or pEGFP-C3 and challenge with HIV-1 89.6 . Results are shown as FFU/ml and are mean ± s.d. of a representative experiment performed in duplicate. (C) Transient over-expression of REAF-EGFP in HeLa-CD4 cells results in decreased RU5 and late RT products 8 hr post challenge with HIV-1 89.6 . HIV-1 DNA copies are normalised to genomic GAPDH and presented per million cells. Results are mean ± s.d. of a representative experiment performed in duplicate. (D) Restriction by REAF is dependent upon viral route of entry. HIV-1 89.6Δenv was pseudotyped with VSV-G envelope and used to challenge HeLa-CD4 cells following REAF siRNA knockdown. p24 immunostaining was used to detect FFU compared with HIV-1 89.6 . Results are shown as FFU/ml and are mean ± s.d. of a representative experiment performed in duplicate.

Techniques Used: Over Expression, Transfection, Western Blot, Expressing, Infection, Immunostaining

REAF interacts with viral nucleic acids. (A) Oligo d(T) immunoprecipitation of HeLa-CD4 cell lysate shows that REAF associates with captured RNA. Cell lysate was treated with DNase or titrated RNaseA/H before incubation with oligo (dT) beads. Immunoprecipitated protein was analysed by Western blotting and probed for REAF along with positive (PABP) and negative (GAPDH) controls. (B) HEK 293 T cells were transiently transfected with pEGFP-C3 or REAF-EGFP and the Western blot probed with α-REAF antibody. Endogenous and exogenous REAF are detectable in input samples (lanes 1 and 2) and following IP of samples transfected with pEGFP-C3 (endogenous REAF; lanes 7 and 8) or REAF-EGFP (endogenous and exogenous REAF; lane 9 and 10) with α-REAF antibody, but not after IP with IgG alone (lanes 3–6). (C) The amount of RU5 or late HIV-1 DNA qPCR product is quantified and normalised to input and IgG negative controls. Endogenous REAF associates with viral nucleic acids and this is enriched in the cells over-expressing REAF-EGFP.
Figure Legend Snippet: REAF interacts with viral nucleic acids. (A) Oligo d(T) immunoprecipitation of HeLa-CD4 cell lysate shows that REAF associates with captured RNA. Cell lysate was treated with DNase or titrated RNaseA/H before incubation with oligo (dT) beads. Immunoprecipitated protein was analysed by Western blotting and probed for REAF along with positive (PABP) and negative (GAPDH) controls. (B) HEK 293 T cells were transiently transfected with pEGFP-C3 or REAF-EGFP and the Western blot probed with α-REAF antibody. Endogenous and exogenous REAF are detectable in input samples (lanes 1 and 2) and following IP of samples transfected with pEGFP-C3 (endogenous REAF; lanes 7 and 8) or REAF-EGFP (endogenous and exogenous REAF; lane 9 and 10) with α-REAF antibody, but not after IP with IgG alone (lanes 3–6). (C) The amount of RU5 or late HIV-1 DNA qPCR product is quantified and normalised to input and IgG negative controls. Endogenous REAF associates with viral nucleic acids and this is enriched in the cells over-expressing REAF-EGFP.

Techniques Used: Immunoprecipitation, Incubation, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Expressing

7) Product Images from "Novel restriction factor RNA-associated early-stage anti-viral factor (REAF) inhibits human and simian immunodeficiency viruses"

Article Title: Novel restriction factor RNA-associated early-stage anti-viral factor (REAF) inhibits human and simian immunodeficiency viruses

Journal: Retrovirology

doi: 10.1186/1742-4690-11-3

Over-expression of REAF confers restriction which is viral route of entry dependent. (A) Over-expression of REAF restricts early HIV-1 DNA production. HEK 293 T cells were transiently transfected with pEGFP-C3 or REAF-EGFP and the Western blot probed with α-GFP antibody. Transfection of REAF-EGFP results in expression of REAF (110, 160 and 250 kDa including EGFP tag (30 kDa). Endogenous REAF was knocked out of HeLa-CD4 cells using siRNA targeting its 3’ UTR. (B) Infected cells were measured following transient over-expression of REAF-EGFP or pEGFP-C3 and challenge with HIV-1 89.6 . Results are shown as FFU/ml and are mean ± s.d. of a representative experiment performed in duplicate. (C) Transient over-expression of REAF-EGFP in HeLa-CD4 cells results in decreased RU5 and late RT products 8 hr post challenge with HIV-1 89.6 . HIV-1 DNA copies are normalised to genomic GAPDH and presented per million cells. Results are mean ± s.d. of a representative experiment performed in duplicate. (D) Restriction by REAF is dependent upon viral route of entry. HIV-1 89.6Δenv was pseudotyped with VSV-G envelope and used to challenge HeLa-CD4 cells following REAF siRNA knockdown. p24 immunostaining was used to detect FFU compared with HIV-1 89.6 . Results are shown as FFU/ml and are mean ± s.d. of a representative experiment performed in duplicate.
Figure Legend Snippet: Over-expression of REAF confers restriction which is viral route of entry dependent. (A) Over-expression of REAF restricts early HIV-1 DNA production. HEK 293 T cells were transiently transfected with pEGFP-C3 or REAF-EGFP and the Western blot probed with α-GFP antibody. Transfection of REAF-EGFP results in expression of REAF (110, 160 and 250 kDa including EGFP tag (30 kDa). Endogenous REAF was knocked out of HeLa-CD4 cells using siRNA targeting its 3’ UTR. (B) Infected cells were measured following transient over-expression of REAF-EGFP or pEGFP-C3 and challenge with HIV-1 89.6 . Results are shown as FFU/ml and are mean ± s.d. of a representative experiment performed in duplicate. (C) Transient over-expression of REAF-EGFP in HeLa-CD4 cells results in decreased RU5 and late RT products 8 hr post challenge with HIV-1 89.6 . HIV-1 DNA copies are normalised to genomic GAPDH and presented per million cells. Results are mean ± s.d. of a representative experiment performed in duplicate. (D) Restriction by REAF is dependent upon viral route of entry. HIV-1 89.6Δenv was pseudotyped with VSV-G envelope and used to challenge HeLa-CD4 cells following REAF siRNA knockdown. p24 immunostaining was used to detect FFU compared with HIV-1 89.6 . Results are shown as FFU/ml and are mean ± s.d. of a representative experiment performed in duplicate.

Techniques Used: Over Expression, Transfection, Western Blot, Expressing, Infection, Immunostaining

REAF interacts with viral nucleic acids. (A) Oligo d(T) immunoprecipitation of HeLa-CD4 cell lysate shows that REAF associates with captured RNA. Cell lysate was treated with DNase or titrated RNaseA/H before incubation with oligo (dT) beads. Immunoprecipitated protein was analysed by Western blotting and probed for REAF along with positive (PABP) and negative (GAPDH) controls. (B) HEK 293 T cells were transiently transfected with pEGFP-C3 or REAF-EGFP and the Western blot probed with α-REAF antibody. Endogenous and exogenous REAF are detectable in input samples (lanes 1 and 2) and following IP of samples transfected with pEGFP-C3 (endogenous REAF; lanes 7 and 8) or REAF-EGFP (endogenous and exogenous REAF; lane 9 and 10) with α-REAF antibody, but not after IP with IgG alone (lanes 3–6). (C) The amount of RU5 or late HIV-1 DNA qPCR product is quantified and normalised to input and IgG negative controls. Endogenous REAF associates with viral nucleic acids and this is enriched in the cells over-expressing REAF-EGFP.
Figure Legend Snippet: REAF interacts with viral nucleic acids. (A) Oligo d(T) immunoprecipitation of HeLa-CD4 cell lysate shows that REAF associates with captured RNA. Cell lysate was treated with DNase or titrated RNaseA/H before incubation with oligo (dT) beads. Immunoprecipitated protein was analysed by Western blotting and probed for REAF along with positive (PABP) and negative (GAPDH) controls. (B) HEK 293 T cells were transiently transfected with pEGFP-C3 or REAF-EGFP and the Western blot probed with α-REAF antibody. Endogenous and exogenous REAF are detectable in input samples (lanes 1 and 2) and following IP of samples transfected with pEGFP-C3 (endogenous REAF; lanes 7 and 8) or REAF-EGFP (endogenous and exogenous REAF; lane 9 and 10) with α-REAF antibody, but not after IP with IgG alone (lanes 3–6). (C) The amount of RU5 or late HIV-1 DNA qPCR product is quantified and normalised to input and IgG negative controls. Endogenous REAF associates with viral nucleic acids and this is enriched in the cells over-expressing REAF-EGFP.

Techniques Used: Immunoprecipitation, Incubation, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Expressing

8) Product Images from "Mycobacterial and HIV Infections Up-Regulated Human Zinc Finger Protein 134, a Novel Positive Regulator of HIV-1 LTR Activity and Viral Propagation"

Article Title: Mycobacterial and HIV Infections Up-Regulated Human Zinc Finger Protein 134, a Novel Positive Regulator of HIV-1 LTR Activity and Viral Propagation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0104908

hZNF-134 over expression enhanced the transcriptional potential of LTR and increased viral titers. (A) Luciferase activity was measured in the presence or absence of Tat following transfection with pEGFP-C3 or ZNF-134-GFP-C3 vector into HEK293T cells along with the reporter construct, pLTR-luc. Luciferase activity was measured in terms of relative luminescence units (RLU) from HEK293T cell lysates after 48 hours and plotted as fold increase in the luciferase activity. Cells over-expressing hZNF-134-GFP showed increased luciferase activity when compared to GFP alone. (B) pNL4-3 was transfected in HEK293T cells with either GFP or hZNF-134-GFP. After 48 hours, the viral transcription was measured by qRT-PCR. The plot shows that viral transcripts increased in the presence of over-expressed hZNF-134. (C) HEK293T cells over-expressing either GFP or hZNF-134-GFP were transfected with pNL4-3 vector and after 48 hours scored for viral p24 by ELISA. Inset shows the Western blot confirming the over-expression of either GFP or hZNF-134-GFP in HEK293T cells. For loading control, expression of GAPDH was detected by Western blots in both the cases. Over-expression of hZNF-134 increased viral titers. All the transfection efficiencies were normalized to pRL-TK transfections. All experiments were done more than three times and error bars represents mean ± SD. Student's t -test was performed and *p
Figure Legend Snippet: hZNF-134 over expression enhanced the transcriptional potential of LTR and increased viral titers. (A) Luciferase activity was measured in the presence or absence of Tat following transfection with pEGFP-C3 or ZNF-134-GFP-C3 vector into HEK293T cells along with the reporter construct, pLTR-luc. Luciferase activity was measured in terms of relative luminescence units (RLU) from HEK293T cell lysates after 48 hours and plotted as fold increase in the luciferase activity. Cells over-expressing hZNF-134-GFP showed increased luciferase activity when compared to GFP alone. (B) pNL4-3 was transfected in HEK293T cells with either GFP or hZNF-134-GFP. After 48 hours, the viral transcription was measured by qRT-PCR. The plot shows that viral transcripts increased in the presence of over-expressed hZNF-134. (C) HEK293T cells over-expressing either GFP or hZNF-134-GFP were transfected with pNL4-3 vector and after 48 hours scored for viral p24 by ELISA. Inset shows the Western blot confirming the over-expression of either GFP or hZNF-134-GFP in HEK293T cells. For loading control, expression of GAPDH was detected by Western blots in both the cases. Over-expression of hZNF-134 increased viral titers. All the transfection efficiencies were normalized to pRL-TK transfections. All experiments were done more than three times and error bars represents mean ± SD. Student's t -test was performed and *p

Techniques Used: Over Expression, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Construct, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot

hZNF-134-GFP is localized in the nucleus of HEK293T and Astrocytoma 1321N1 cells. HEK293T and 1321N1 cells were transfected with expression plasmids pEGFP-C3 or ZNF-134-GFP-C3. The cells were fixed after 48 hours and visualized under confocal microscopy. Panels show Trans, DAPI, GFP, and Merged images. hZNF-134-GFP protein was localized in the nucleus of HEK293T and 1321N1 cells whereas GFP control showed both nuclear and cytoplasmic localization. Localization was confirmed by 3 independent experiments.
Figure Legend Snippet: hZNF-134-GFP is localized in the nucleus of HEK293T and Astrocytoma 1321N1 cells. HEK293T and 1321N1 cells were transfected with expression plasmids pEGFP-C3 or ZNF-134-GFP-C3. The cells were fixed after 48 hours and visualized under confocal microscopy. Panels show Trans, DAPI, GFP, and Merged images. hZNF-134-GFP protein was localized in the nucleus of HEK293T and 1321N1 cells whereas GFP control showed both nuclear and cytoplasmic localization. Localization was confirmed by 3 independent experiments.

Techniques Used: Transfection, Expressing, Confocal Microscopy

9) Product Images from "PKC-? Promotes Renal Tubular Cell Apoptosis Associated with Proteinuria"

Article Title: PKC-? Promotes Renal Tubular Cell Apoptosis Associated with Proteinuria

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2009070760

Dominant negative PKC suppresses albumin-induced RPTC apoptosis. RPTCs were cotransfected with pEGFP-C3 and dominant-negative PKC-δ (dn-PKC-δ), dominant-negative PKC-α (dn-PKC-α), or empty pcDNA3.1 vector. The cells were then incubated for 24 hours with 20 mg/ml albumin and stained with Hoechst 33342 for morphologic examination of apoptosis. (A) Representative cell morphology. Magnification, ×400. Arrows show transfected cells that showed typical apoptotic morphology. (B) Percentage of apoptosis in GFP-labeled transfected cells. Data are mean ± SD, n = 3. h P
Figure Legend Snippet: Dominant negative PKC suppresses albumin-induced RPTC apoptosis. RPTCs were cotransfected with pEGFP-C3 and dominant-negative PKC-δ (dn-PKC-δ), dominant-negative PKC-α (dn-PKC-α), or empty pcDNA3.1 vector. The cells were then incubated for 24 hours with 20 mg/ml albumin and stained with Hoechst 33342 for morphologic examination of apoptosis. (A) Representative cell morphology. Magnification, ×400. Arrows show transfected cells that showed typical apoptotic morphology. (B) Percentage of apoptosis in GFP-labeled transfected cells. Data are mean ± SD, n = 3. h P

Techniques Used: Dominant Negative Mutation, Plasmid Preparation, Incubation, Staining, Transfection, Labeling

10) Product Images from "Protective effects of tetramethylpyrazine on rat retinal cell cultures"

Article Title: Protective effects of tetramethylpyrazine on rat retinal cell cultures

Journal: Neurochemistry international

doi: 10.1016/j.neuint.2007.12.008

TMP inhibits ROS-induced downregulation of rattin to promote retinal cell survival (A-F) Cytoplasmic immunoreactivity of rattin (green) in both MAP-2-positive neurons (red) and other non-neuronal cells in retinal cell cultures treated with vehicles (A B), 10 μM H 2 O 2 alone (C D), or “10 μM H 2 O 2 +50 μM TMP” (E F). Arrows in merged image D indicate rattin-negative neurons. (G) Changes in abundance of rattin protein with cultivation time or indicated treatments by Western blots. The results were quantified by optical densitometry and are depicted relative to the 1-week old controls. (H) Expression of rattin mRNA in cultures following indicated treatments was examined by RT-PCR. The results are depicted relative to levels in vehicle-treated cells (H 2 O 2 = 0; TMP = 0). (I-T) siRNA mediated downregulation of rattin associated with retinal cell death visualized by fluorescence microscopy. Cells were co-transfected with both pEGFP-C3 and either the control siRNA (I-K, O-Q) or rattin siRNA (L-N, R-T). Immunocytochemistry shows that most GFP-positive cells are rattin-immunoreactive in controls (J-K, arrows) but demonstrate remarkably decreased immunoreactivity of rattin in cells 30 hr after rattin siRNA transfection (L-N, arrows). TUNEL staining (red) demonstrates no evident cell death in control (O-Q) and extensive cell damage in most GFP-positive cells (R-T, arrows). (U) Abundance of rattin in cells 30 hr after transfection by Western blots. UT = untransfected. (V) Quantification of TUNEL-positive cells in cells 30 hr after transfection. β-actin was used as loading controls. Bars depict mean ± S.E.M., ** p
Figure Legend Snippet: TMP inhibits ROS-induced downregulation of rattin to promote retinal cell survival (A-F) Cytoplasmic immunoreactivity of rattin (green) in both MAP-2-positive neurons (red) and other non-neuronal cells in retinal cell cultures treated with vehicles (A B), 10 μM H 2 O 2 alone (C D), or “10 μM H 2 O 2 +50 μM TMP” (E F). Arrows in merged image D indicate rattin-negative neurons. (G) Changes in abundance of rattin protein with cultivation time or indicated treatments by Western blots. The results were quantified by optical densitometry and are depicted relative to the 1-week old controls. (H) Expression of rattin mRNA in cultures following indicated treatments was examined by RT-PCR. The results are depicted relative to levels in vehicle-treated cells (H 2 O 2 = 0; TMP = 0). (I-T) siRNA mediated downregulation of rattin associated with retinal cell death visualized by fluorescence microscopy. Cells were co-transfected with both pEGFP-C3 and either the control siRNA (I-K, O-Q) or rattin siRNA (L-N, R-T). Immunocytochemistry shows that most GFP-positive cells are rattin-immunoreactive in controls (J-K, arrows) but demonstrate remarkably decreased immunoreactivity of rattin in cells 30 hr after rattin siRNA transfection (L-N, arrows). TUNEL staining (red) demonstrates no evident cell death in control (O-Q) and extensive cell damage in most GFP-positive cells (R-T, arrows). (U) Abundance of rattin in cells 30 hr after transfection by Western blots. UT = untransfected. (V) Quantification of TUNEL-positive cells in cells 30 hr after transfection. β-actin was used as loading controls. Bars depict mean ± S.E.M., ** p

Techniques Used: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Microscopy, Transfection, Immunocytochemistry, TUNEL Assay, Staining

11) Product Images from "The apoptotic response in HCT116BAX-/- cancer cells becomes rapidly saturated with increasing expression of a GFP-BAX fusion protein"

Article Title: The apoptotic response in HCT116BAX-/- cancer cells becomes rapidly saturated with increasing expression of a GFP-BAX fusion protein

Journal: BMC Cancer

doi: 10.1186/1471-2407-10-554

Transient transfection of GFP-BAX fusion protein into HCT116 BAX -/- cells rescues cell death after Indomethacin treatment . (A) HCT116 BAX-/- cells are resistant to indomethacin. Line graph showing percentage of apoptotic cells in the medium after exposure of 500 μM indomethacin for wild type (WT) and BAX -deficient HCT116 cells over time. (B) Rescue of the cell death phenotype by transfection of an exogenous GFP- Bax gene. HCT116 BAX -/- cells were transfected with either no plasmid, pEGFP-C3 (GFP), or pEGFP-C3-BAX (GFP-BAX), which expresses a fusion protein of GFP in phase with the murine Bax coding region under the control of the CMV immediate early promoter. Twenty-four hours after transfection, cells were treated with 500 μM indomethacin (+Indo) or vehicle (-Indo). Transfection efficiency was ~60% in each condition, based on the proportion of cells positive for GFP expression. Forty-eight hours after indomethacin treatment, cell death was assessed. Transfection of GFP-BAX into cells that were later treated with indomethacin caused a significant increase in death over GFP transfection and indomethacin-treated cells ( t -test, * P = 0.0001). Values shown are mean ± SD in both graphs of data collected from 3 independent experiments.
Figure Legend Snippet: Transient transfection of GFP-BAX fusion protein into HCT116 BAX -/- cells rescues cell death after Indomethacin treatment . (A) HCT116 BAX-/- cells are resistant to indomethacin. Line graph showing percentage of apoptotic cells in the medium after exposure of 500 μM indomethacin for wild type (WT) and BAX -deficient HCT116 cells over time. (B) Rescue of the cell death phenotype by transfection of an exogenous GFP- Bax gene. HCT116 BAX -/- cells were transfected with either no plasmid, pEGFP-C3 (GFP), or pEGFP-C3-BAX (GFP-BAX), which expresses a fusion protein of GFP in phase with the murine Bax coding region under the control of the CMV immediate early promoter. Twenty-four hours after transfection, cells were treated with 500 μM indomethacin (+Indo) or vehicle (-Indo). Transfection efficiency was ~60% in each condition, based on the proportion of cells positive for GFP expression. Forty-eight hours after indomethacin treatment, cell death was assessed. Transfection of GFP-BAX into cells that were later treated with indomethacin caused a significant increase in death over GFP transfection and indomethacin-treated cells ( t -test, * P = 0.0001). Values shown are mean ± SD in both graphs of data collected from 3 independent experiments.

Techniques Used: Transfection, Plasmid Preparation, Expressing

12) Product Images from "Ankyrin-B Regulates Cav2.1 and Cav2.2 Channel Expression and Targeting *"

Article Title: Ankyrin-B Regulates Cav2.1 and Cav2.2 Channel Expression and Targeting *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.523639

Ca v 2. 1/Ca v 2.2 tyrosine residues in ABM are critical for cellular targeting and ankyrin interaction. A , HEK293 cells transfected with pEGFP-C3 demonstrated a primarily nuclear localization pattern. B and D , in contrast, HEK293 cells transfected with full-length
Figure Legend Snippet: Ca v 2. 1/Ca v 2.2 tyrosine residues in ABM are critical for cellular targeting and ankyrin interaction. A , HEK293 cells transfected with pEGFP-C3 demonstrated a primarily nuclear localization pattern. B and D , in contrast, HEK293 cells transfected with full-length

Techniques Used: Transfection

Ca v 2.1/Ca v 2.2 tyrosine residues in ABM are critical for cellular targeting. HEK293 cells transfected with pEGFP-C3 demonstrated a primarily nuclear localization pattern ( A and D ), whereas HEK293 cells transfected with Ca v 2.1 DII/III-GFP ( B ) or Ca v 2.2
Figure Legend Snippet: Ca v 2.1/Ca v 2.2 tyrosine residues in ABM are critical for cellular targeting. HEK293 cells transfected with pEGFP-C3 demonstrated a primarily nuclear localization pattern ( A and D ), whereas HEK293 cells transfected with Ca v 2.1 DII/III-GFP ( B ) or Ca v 2.2

Techniques Used: Transfection

13) Product Images from "Identification of Nuclear Localization Signals in the ORF2 Protein of Porcine Circovirus Type 3"

Article Title: Identification of Nuclear Localization Signals in the ORF2 Protein of Porcine Circovirus Type 3

Journal: Viruses

doi: 10.3390/v11121086

The key amino acids of NLS in ORF2-1-3. ( A ) Mutants of ORF2-1-3 gene were inserted into pEGFP-C3, and the key amino acids predicted in ORF2-1-3 are listed by underline. ( B ) PK-15 cells were transfected with mutant plasmids, and the localization of fusion proteins was observed by confocal microscopy. Scale bars = 20 μm. ( C ) and ( D ) After 24 h, the nucleus and cytoplasm in transfected cells were extracted and the expressed proteins were determined by western blot. Nuclear/cytoplasmic distribution of the expressed proteins was further analyzed through densitometric quantification using Image-Pro software, data from three independent experiments are shown on the graph as the average ± standard error. One-way ANOVA; ** p
Figure Legend Snippet: The key amino acids of NLS in ORF2-1-3. ( A ) Mutants of ORF2-1-3 gene were inserted into pEGFP-C3, and the key amino acids predicted in ORF2-1-3 are listed by underline. ( B ) PK-15 cells were transfected with mutant plasmids, and the localization of fusion proteins was observed by confocal microscopy. Scale bars = 20 μm. ( C ) and ( D ) After 24 h, the nucleus and cytoplasm in transfected cells were extracted and the expressed proteins were determined by western blot. Nuclear/cytoplasmic distribution of the expressed proteins was further analyzed through densitometric quantification using Image-Pro software, data from three independent experiments are shown on the graph as the average ± standard error. One-way ANOVA; ** p

Techniques Used: Transfection, Mutagenesis, Confocal Microscopy, Western Blot, Software

The main nucleolar localization signal motifs in ORF2-1-2. ( A ) Mutants of ORF2-1-2 gene were inserted into pEGFP-C3 and predicted nucleolar localization signal motifs are underlined. ( B ) PK-15 cells were transfected with plasmids, after 24 h, the cells were fixed and the localization of fusion proteins was observed by confocal microscopy. Scale bars = 20 μm.
Figure Legend Snippet: The main nucleolar localization signal motifs in ORF2-1-2. ( A ) Mutants of ORF2-1-2 gene were inserted into pEGFP-C3 and predicted nucleolar localization signal motifs are underlined. ( B ) PK-15 cells were transfected with plasmids, after 24 h, the cells were fixed and the localization of fusion proteins was observed by confocal microscopy. Scale bars = 20 μm.

Techniques Used: Transfection, Confocal Microscopy

14) Product Images from "Identification of Nuclear Localization Signals in the ORF2 Protein of Porcine Circovirus Type 3"

Article Title: Identification of Nuclear Localization Signals in the ORF2 Protein of Porcine Circovirus Type 3

Journal: Viruses

doi: 10.3390/v11121086

The key amino acids of NLS in ORF2-1-3. ( A ) Mutants of ORF2-1-3 gene were inserted into pEGFP-C3, and the key amino acids predicted in ORF2-1-3 are listed by underline. ( B ) PK-15 cells were transfected with mutant plasmids, and the localization of fusion proteins was observed by confocal microscopy. Scale bars = 20 μm. ( C ) and ( D ) After 24 h, the nucleus and cytoplasm in transfected cells were extracted and the expressed proteins were determined by western blot. Nuclear/cytoplasmic distribution of the expressed proteins was further analyzed through densitometric quantification using Image-Pro software, data from three independent experiments are shown on the graph as the average ± standard error. One-way ANOVA; ** p
Figure Legend Snippet: The key amino acids of NLS in ORF2-1-3. ( A ) Mutants of ORF2-1-3 gene were inserted into pEGFP-C3, and the key amino acids predicted in ORF2-1-3 are listed by underline. ( B ) PK-15 cells were transfected with mutant plasmids, and the localization of fusion proteins was observed by confocal microscopy. Scale bars = 20 μm. ( C ) and ( D ) After 24 h, the nucleus and cytoplasm in transfected cells were extracted and the expressed proteins were determined by western blot. Nuclear/cytoplasmic distribution of the expressed proteins was further analyzed through densitometric quantification using Image-Pro software, data from three independent experiments are shown on the graph as the average ± standard error. One-way ANOVA; ** p

Techniques Used: Transfection, Mutagenesis, Confocal Microscopy, Western Blot, Software

The main nucleolar localization signal motifs in ORF2-1-2. ( A ) Mutants of ORF2-1-2 gene were inserted into pEGFP-C3 and predicted nucleolar localization signal motifs are underlined. ( B ) PK-15 cells were transfected with plasmids, after 24 h, the cells were fixed and the localization of fusion proteins was observed by confocal microscopy. Scale bars = 20 μm.
Figure Legend Snippet: The main nucleolar localization signal motifs in ORF2-1-2. ( A ) Mutants of ORF2-1-2 gene were inserted into pEGFP-C3 and predicted nucleolar localization signal motifs are underlined. ( B ) PK-15 cells were transfected with plasmids, after 24 h, the cells were fixed and the localization of fusion proteins was observed by confocal microscopy. Scale bars = 20 μm.

Techniques Used: Transfection, Confocal Microscopy

15) Product Images from "Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice"

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

Journal: Endocrinology

doi: 10.1210/en.2015-1556

Determination of the control strength of ADAM17 on ACE2 activity. 832/13 cells were cotransfected with 25 ng/well mACE2/pcDNA3.1 and 2 μg/well of a mix of pAd17 and pAd17E406A. The pAd17 content in the mix were 0, 0.4, 0.8, 1.2, 1.6, and 2 μg/well in four experiments and 0, 0.05, 0.1, 0.2, 0,4, and 2 μg/well in eight experiments. ADAM17 activities (A) and ACE2 activities (B) were measured. C, For estimation of transfection efficiencies, green fluorescent 832/13 cells transfected with 2 μg pEGFP-C3 and 25 ng mACE/pcDNA3.1 were determined as in the upper right panel compared with control cells in the upper left panel. To estimate cotransfection efficiency, cells cotransfected with a plasmid encoding the red fluorescent tdTomato and pAd17 (lower left panel) were compared to cells cotransfected with plasmids for eGFP and tdTomato expression (lower right panel). For the different amounts of pAd17 used in the transfection experiments, shed ACE2 (D) and cellular ACE2 (E) were plotted against the estimated ADAM17 activity of the transfected cells, ie, corrected for the transfection efficiency. Curves describing saturation kinetics were fitted to the data. F, Based on the model fits, the control strengths were calculated and plotted as a function of the ADAM17 activity of transfected cells.
Figure Legend Snippet: Determination of the control strength of ADAM17 on ACE2 activity. 832/13 cells were cotransfected with 25 ng/well mACE2/pcDNA3.1 and 2 μg/well of a mix of pAd17 and pAd17E406A. The pAd17 content in the mix were 0, 0.4, 0.8, 1.2, 1.6, and 2 μg/well in four experiments and 0, 0.05, 0.1, 0.2, 0,4, and 2 μg/well in eight experiments. ADAM17 activities (A) and ACE2 activities (B) were measured. C, For estimation of transfection efficiencies, green fluorescent 832/13 cells transfected with 2 μg pEGFP-C3 and 25 ng mACE/pcDNA3.1 were determined as in the upper right panel compared with control cells in the upper left panel. To estimate cotransfection efficiency, cells cotransfected with a plasmid encoding the red fluorescent tdTomato and pAd17 (lower left panel) were compared to cells cotransfected with plasmids for eGFP and tdTomato expression (lower right panel). For the different amounts of pAd17 used in the transfection experiments, shed ACE2 (D) and cellular ACE2 (E) were plotted against the estimated ADAM17 activity of the transfected cells, ie, corrected for the transfection efficiency. Curves describing saturation kinetics were fitted to the data. F, Based on the model fits, the control strengths were calculated and plotted as a function of the ADAM17 activity of transfected cells.

Techniques Used: Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Expressing

16) Product Images from "Green Fluorescent Protein-Tagged Adeno-Associated Virus Particles Allow the Study of Cytosolic and Nuclear Trafficking †"

Article Title: Green Fluorescent Protein-Tagged Adeno-Associated Virus Particles Allow the Study of Cytosolic and Nuclear Trafficking †

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11776-11787.2005

Schematic representation of the plasmids. (A) The plasmid pGFP-VP2 encodes the GFP-VP2 fusion protein. VP2 was amplified by PCR from pUC-AV2 and cloned into the multiple cloning site of pEGFP-C3 (Clontech). During this step, the VP2 start codon was deleted. (B) To produce wild-type AAV, the plasmid pUC-AV2 was used (top). A G-to-C substitution within the wobble position of the VP2 start codon (T138) was introduced, resulting in the plasmid pUC-AV2-VP2k.o. (bottom). Due to the substitution, VP2 expression was abolished without altering the amino acid sequence of VP1.
Figure Legend Snippet: Schematic representation of the plasmids. (A) The plasmid pGFP-VP2 encodes the GFP-VP2 fusion protein. VP2 was amplified by PCR from pUC-AV2 and cloned into the multiple cloning site of pEGFP-C3 (Clontech). During this step, the VP2 start codon was deleted. (B) To produce wild-type AAV, the plasmid pUC-AV2 was used (top). A G-to-C substitution within the wobble position of the VP2 start codon (T138) was introduced, resulting in the plasmid pUC-AV2-VP2k.o. (bottom). Due to the substitution, VP2 expression was abolished without altering the amino acid sequence of VP1.

Techniques Used: Plasmid Preparation, Amplification, Polymerase Chain Reaction, Clone Assay, Expressing, Sequencing

17) Product Images from "ATM regulates Mre11-dependent DNA end-degradation and microhomology-mediated end joining"

Article Title: ATM regulates Mre11-dependent DNA end-degradation and microhomology-mediated end joining

Journal: Cell Cycle

doi: 10.4161/cc.9.14.12363

Western immunoblot for Mre11 and EGFP after MMEJ repair in Mirin-treated or Mre11-knockdown cells. WI-38VA13 (CF) and AT5BIVA (AT) cells were transfected with either linear or circular pMMEJ. Repair was allowed and cell extracts were then prepared. Cells treated with 25 mM Mirin or in which Mre11 was knocked down by transduction with lentivirus encoding Mre11 shRNA (Mre11 sh3 and Mre11 sh4) were also analyzed. Cells transduced with empty vector (Lanes 2 and 9) or virus-free transduction medium (Lanes 4 and 10) were included as controls. Transfection with linear pEGFP-c3 was used as an EGFP expression control (Lanes 1 and 7). Cell extracts were subjected to SDS-PAGE, transferred to a membrane and then probed for Mre11 and EGFP. G6PD was assayed for as a loading control.
Figure Legend Snippet: Western immunoblot for Mre11 and EGFP after MMEJ repair in Mirin-treated or Mre11-knockdown cells. WI-38VA13 (CF) and AT5BIVA (AT) cells were transfected with either linear or circular pMMEJ. Repair was allowed and cell extracts were then prepared. Cells treated with 25 mM Mirin or in which Mre11 was knocked down by transduction with lentivirus encoding Mre11 shRNA (Mre11 sh3 and Mre11 sh4) were also analyzed. Cells transduced with empty vector (Lanes 2 and 9) or virus-free transduction medium (Lanes 4 and 10) were included as controls. Transfection with linear pEGFP-c3 was used as an EGFP expression control (Lanes 1 and 7). Cell extracts were subjected to SDS-PAGE, transferred to a membrane and then probed for Mre11 and EGFP. G6PD was assayed for as a loading control.

Techniques Used: Western Blot, Transfection, Transduction, shRNA, Plasmid Preparation, Expressing, SDS Page

Suppression of in vivo MMEJ after Mirin-treatment or knocking down Mre11. (A) Schematic representation of the pMMEJ plasmid used to assess in vivo MMEJ repair. The pMMEJ plasmid was derived from the pEGFP-C3 plasmid by an insertion of 35 bp within the ORF of the wtEGFP gene. This creates an I- Sce I megaendonuclease recognition site flanked by two 5 bp microhomologies. The repair of a linearized pMMEJ plasmid by MMEJ reconstitutes the wtEGFP gene and allows expression of EGFP. (B) WI-38VA13 (CF) and AT5BIVA (AT) cells were transfected with either linear or circular pMMEJ and with a mCherry transfection control plasmid. Repair was allowed and cells were analyzed for fluorescent protein expression by flow cytometry. Cells treated with 25 mM Mirin or in which Mre11 was knocked down by transduction with lentivirus encoding Mre11 shRNA (Mre11 sh3 or Mre11 sh4) were also analyzed. Cells transduced with empty vector or virus-free transduction medium were included as controls. Repair through MMEJ is represented by %EGFP expression; %EGFP expression = (number of cells expressing both EGFP and mCherry/number of cells expressing mCherry) × 100.
Figure Legend Snippet: Suppression of in vivo MMEJ after Mirin-treatment or knocking down Mre11. (A) Schematic representation of the pMMEJ plasmid used to assess in vivo MMEJ repair. The pMMEJ plasmid was derived from the pEGFP-C3 plasmid by an insertion of 35 bp within the ORF of the wtEGFP gene. This creates an I- Sce I megaendonuclease recognition site flanked by two 5 bp microhomologies. The repair of a linearized pMMEJ plasmid by MMEJ reconstitutes the wtEGFP gene and allows expression of EGFP. (B) WI-38VA13 (CF) and AT5BIVA (AT) cells were transfected with either linear or circular pMMEJ and with a mCherry transfection control plasmid. Repair was allowed and cells were analyzed for fluorescent protein expression by flow cytometry. Cells treated with 25 mM Mirin or in which Mre11 was knocked down by transduction with lentivirus encoding Mre11 shRNA (Mre11 sh3 or Mre11 sh4) were also analyzed. Cells transduced with empty vector or virus-free transduction medium were included as controls. Repair through MMEJ is represented by %EGFP expression; %EGFP expression = (number of cells expressing both EGFP and mCherry/number of cells expressing mCherry) × 100.

Techniques Used: In Vivo, Plasmid Preparation, Derivative Assay, Expressing, Transfection, Flow Cytometry, Cytometry, Transduction, shRNA

18) Product Images from "Intramembrane Proteolysis and Endoplasmic Reticulum Retention of Hepatitis C Virus Core Protein"

Article Title: Intramembrane Proteolysis and Endoplasmic Reticulum Retention of Hepatitis C Virus Core Protein

Journal: Journal of Virology

doi: 10.1128/JVI.78.12.6370-6380.2004

Expression plasmids used in this study. The genes encoding HCV proteins and their mutants were cloned into pcDNA3.1FlagHA, pcDNA3.1/ myc -His C, or pEGFP-C3 as described in Materials and Methods. Other plasmids are described in the text or in the other figure legends.
Figure Legend Snippet: Expression plasmids used in this study. The genes encoding HCV proteins and their mutants were cloned into pcDNA3.1FlagHA, pcDNA3.1/ myc -His C, or pEGFP-C3 as described in Materials and Methods. Other plasmids are described in the text or in the other figure legends.

Techniques Used: Expressing, Clone Assay

19) Product Images from "Intramembrane Proteolysis and Endoplasmic Reticulum Retention of Hepatitis C Virus Core Protein"

Article Title: Intramembrane Proteolysis and Endoplasmic Reticulum Retention of Hepatitis C Virus Core Protein

Journal: Journal of Virology

doi: 10.1128/JVI.78.12.6370-6380.2004

Expression plasmids used in this study. The genes encoding HCV proteins and their mutants were cloned into pcDNA3.1FlagHA, pcDNA3.1/ myc -His C, or pEGFP-C3 as described in Materials and Methods. Other plasmids are described in the text or in the other figure legends.
Figure Legend Snippet: Expression plasmids used in this study. The genes encoding HCV proteins and their mutants were cloned into pcDNA3.1FlagHA, pcDNA3.1/ myc -His C, or pEGFP-C3 as described in Materials and Methods. Other plasmids are described in the text or in the other figure legends.

Techniques Used: Expressing, Clone Assay

20) Product Images from "Novel role of KCNQ2/3 channels in regulating neuronal cell viability"

Article Title: Novel role of KCNQ2/3 channels in regulating neuronal cell viability

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2010.120

Effects of expression of KCNQ2/KCNQ3 channels on caspase-3 activation. ( a – d ) KCNQ2 and KCNQ3 subunits were subcloned into pEGFP-C3 ( a ) and pDsRed2-C1 vectors ( c ) and transfected into CHO cells shown as green and red color in transfected cells
Figure Legend Snippet: Effects of expression of KCNQ2/KCNQ3 channels on caspase-3 activation. ( a – d ) KCNQ2 and KCNQ3 subunits were subcloned into pEGFP-C3 ( a ) and pDsRed2-C1 vectors ( c ) and transfected into CHO cells shown as green and red color in transfected cells

Techniques Used: Expressing, Activation Assay, Transfection

21) Product Images from "Identification of Nuclear Localization Signals in the ORF2 Protein of Porcine Circovirus Type 3"

Article Title: Identification of Nuclear Localization Signals in the ORF2 Protein of Porcine Circovirus Type 3

Journal: Viruses

doi: 10.3390/v11121086

The key amino acids of NLS in ORF2-1-3. ( A ) Mutants of ORF2-1-3 gene were inserted into pEGFP-C3, and the key amino acids predicted in ORF2-1-3 are listed by underline. ( B ) PK-15 cells were transfected with mutant plasmids, and the localization of fusion proteins was observed by confocal microscopy. Scale bars = 20 μm. ( C ) and ( D ) After 24 h, the nucleus and cytoplasm in transfected cells were extracted and the expressed proteins were determined by western blot. Nuclear/cytoplasmic distribution of the expressed proteins was further analyzed through densitometric quantification using Image-Pro software, data from three independent experiments are shown on the graph as the average ± standard error. One-way ANOVA; ** p
Figure Legend Snippet: The key amino acids of NLS in ORF2-1-3. ( A ) Mutants of ORF2-1-3 gene were inserted into pEGFP-C3, and the key amino acids predicted in ORF2-1-3 are listed by underline. ( B ) PK-15 cells were transfected with mutant plasmids, and the localization of fusion proteins was observed by confocal microscopy. Scale bars = 20 μm. ( C ) and ( D ) After 24 h, the nucleus and cytoplasm in transfected cells were extracted and the expressed proteins were determined by western blot. Nuclear/cytoplasmic distribution of the expressed proteins was further analyzed through densitometric quantification using Image-Pro software, data from three independent experiments are shown on the graph as the average ± standard error. One-way ANOVA; ** p

Techniques Used: Transfection, Mutagenesis, Confocal Microscopy, Western Blot, Software

The main nucleolar localization signal motifs in ORF2-1-2. ( A ) Mutants of ORF2-1-2 gene were inserted into pEGFP-C3 and predicted nucleolar localization signal motifs are underlined. ( B ) PK-15 cells were transfected with plasmids, after 24 h, the cells were fixed and the localization of fusion proteins was observed by confocal microscopy. Scale bars = 20 μm.
Figure Legend Snippet: The main nucleolar localization signal motifs in ORF2-1-2. ( A ) Mutants of ORF2-1-2 gene were inserted into pEGFP-C3 and predicted nucleolar localization signal motifs are underlined. ( B ) PK-15 cells were transfected with plasmids, after 24 h, the cells were fixed and the localization of fusion proteins was observed by confocal microscopy. Scale bars = 20 μm.

Techniques Used: Transfection, Confocal Microscopy

22) Product Images from "Ankyrin-B Regulates Cav2.1 and Cav2.2 Channel Expression and Targeting *"

Article Title: Ankyrin-B Regulates Cav2.1 and Cav2.2 Channel Expression and Targeting *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.523639

Ca v 2. 1/Ca v 2.2 tyrosine residues in ABM are critical for cellular targeting and ankyrin interaction. A , HEK293 cells transfected with pEGFP-C3 demonstrated a primarily nuclear localization pattern. B and D , in contrast, HEK293 cells transfected with full-length
Figure Legend Snippet: Ca v 2. 1/Ca v 2.2 tyrosine residues in ABM are critical for cellular targeting and ankyrin interaction. A , HEK293 cells transfected with pEGFP-C3 demonstrated a primarily nuclear localization pattern. B and D , in contrast, HEK293 cells transfected with full-length

Techniques Used: Transfection

Ca v 2.1/Ca v 2.2 tyrosine residues in ABM are critical for cellular targeting. HEK293 cells transfected with pEGFP-C3 demonstrated a primarily nuclear localization pattern ( A and D ), whereas HEK293 cells transfected with Ca v 2.1 DII/III-GFP ( B ) or Ca v 2.2
Figure Legend Snippet: Ca v 2.1/Ca v 2.2 tyrosine residues in ABM are critical for cellular targeting. HEK293 cells transfected with pEGFP-C3 demonstrated a primarily nuclear localization pattern ( A and D ), whereas HEK293 cells transfected with Ca v 2.1 DII/III-GFP ( B ) or Ca v 2.2

Techniques Used: Transfection

23) Product Images from "Full UPF3B function is critical for neuronal differentiation of neural stem cells"

Article Title: Full UPF3B function is critical for neuronal differentiation of neural stem cells

Journal: Molecular Brain

doi: 10.1186/s13041-015-0122-1

Mutations in UPF3B do not affect its cellular distribution in neurons. Neural stem cells were transfected with the pEGFP-C3 expressing GFP only (empty vector) or a pEGFP-C3 derivative expressing one of the UPF3B forms with an N-terminal GFP tag. Cells were diluted and re-plated after 24 h, and then differentiated for 6 days prior to analysis by confocal microscopy. Shown are separate Z-stack projections of GFP and DAPI channels and of the merged channels. The origin region of the enlarged section is indicated. The scale bar represents 20 μm
Figure Legend Snippet: Mutations in UPF3B do not affect its cellular distribution in neurons. Neural stem cells were transfected with the pEGFP-C3 expressing GFP only (empty vector) or a pEGFP-C3 derivative expressing one of the UPF3B forms with an N-terminal GFP tag. Cells were diluted and re-plated after 24 h, and then differentiated for 6 days prior to analysis by confocal microscopy. Shown are separate Z-stack projections of GFP and DAPI channels and of the merged channels. The origin region of the enlarged section is indicated. The scale bar represents 20 μm

Techniques Used: Transfection, Expressing, Plasmid Preparation, Confocal Microscopy

24) Product Images from "Two Potent Nuclear Localization Signals in the Gut-enriched Kr?ppel-like Factor Define a Subfamily of Closely Related Kr?ppel Proteins *"

Article Title: Two Potent Nuclear Localization Signals in the Gut-enriched Kr?ppel-like Factor Define a Subfamily of Closely Related Kr?ppel Proteins *

Journal: The Journal of biological chemistry

doi:

Cellular localization of green fluorescent protein (GFP) fusion proteins a shows the various GFP fusion constructs cloned in the expression vector, pEGFP-C3. The 5′ basic region of GKLF is shown as a filled box and that of zif268/Egr-1 is shown as a shaded box. Construct E is GFP fused to a peptide containing a core NLS within the third zinc finger of zif268/Egr-1. b shows the amino acid sequences of peptides included in constructs C, D , and E . Basic amino acid residues are underlined. c shows the results of fluorescence microscopy of COS-1 cells transfected with the depicted constructs.
Figure Legend Snippet: Cellular localization of green fluorescent protein (GFP) fusion proteins a shows the various GFP fusion constructs cloned in the expression vector, pEGFP-C3. The 5′ basic region of GKLF is shown as a filled box and that of zif268/Egr-1 is shown as a shaded box. Construct E is GFP fused to a peptide containing a core NLS within the third zinc finger of zif268/Egr-1. b shows the amino acid sequences of peptides included in constructs C, D , and E . Basic amino acid residues are underlined. c shows the results of fluorescence microscopy of COS-1 cells transfected with the depicted constructs.

Techniques Used: Construct, Clone Assay, Expressing, Plasmid Preparation, Fluorescence, Microscopy, Transfection

25) Product Images from "Identification of Unusual E6 and E7 Proteins within Avian Papillomaviruses: Cellular Localization, Biophysical Characterization, and Phylogenetic Analysis "

Article Title: Identification of Unusual E6 and E7 Proteins within Avian Papillomaviruses: Cellular Localization, Biophysical Characterization, and Phylogenetic Analysis

Journal: Journal of Virology

doi: 10.1128/JVI.01777-08

Expression of FlPV-1 E6 and FlPV-1 E7 proteins in HaCaT, HeLa, and QT6 cells. Cells were transfected with pEGFP-E6, pEF-myc-E6, pEF-E6-myc, pEGFP-E7, pEF-myc-E7, pEF-E7-myc, and the empty pEGFP-C3 vector. The different E6 and E7 fusion proteins and EGFP
Figure Legend Snippet: Expression of FlPV-1 E6 and FlPV-1 E7 proteins in HaCaT, HeLa, and QT6 cells. Cells were transfected with pEGFP-E6, pEF-myc-E6, pEF-E6-myc, pEGFP-E7, pEF-myc-E7, pEF-E7-myc, and the empty pEGFP-C3 vector. The different E6 and E7 fusion proteins and EGFP

Techniques Used: Expressing, Transfection, Plasmid Preparation

26) Product Images from "Full UPF3B function is critical for neuronal differentiation of neural stem cells"

Article Title: Full UPF3B function is critical for neuronal differentiation of neural stem cells

Journal: Molecular Brain

doi: 10.1186/s13041-015-0122-1

Mutations in UPF3B do not affect its cellular distribution in neurons. Neural stem cells were transfected with the pEGFP-C3 expressing GFP only (empty vector) or a pEGFP-C3 derivative expressing one of the UPF3B forms with an N-terminal GFP tag. Cells were diluted and re-plated after 24 h, and then differentiated for 6 days prior to analysis by confocal microscopy. Shown are separate Z-stack projections of GFP and DAPI channels and of the merged channels. The origin region of the enlarged section is indicated. The scale bar represents 20 μm
Figure Legend Snippet: Mutations in UPF3B do not affect its cellular distribution in neurons. Neural stem cells were transfected with the pEGFP-C3 expressing GFP only (empty vector) or a pEGFP-C3 derivative expressing one of the UPF3B forms with an N-terminal GFP tag. Cells were diluted and re-plated after 24 h, and then differentiated for 6 days prior to analysis by confocal microscopy. Shown are separate Z-stack projections of GFP and DAPI channels and of the merged channels. The origin region of the enlarged section is indicated. The scale bar represents 20 μm

Techniques Used: Transfection, Expressing, Plasmid Preparation, Confocal Microscopy

27) Product Images from "The ESCRT System Is Required for Hepatitis C Virus Production"

Article Title: The ESCRT System Is Required for Hepatitis C Virus Production

Journal: PLoS ONE

doi: 10.1371/journal.pone.0014517

HCV Core interacts with CHMP4b. (A–C) HCV Core colocalizes with CHMP4b. 293FT cells cotransfected with 100 ng of pcDNA3/core (JFH1) and either 100 ng of pCHMP4b-GFP [39] (A) or pFLAG-CHMP4b [39] (B) were examined by confocal laser scanning microscopy. Cells were stained with anti-HCV Core and anti-FLAG polyclonal antibody and were then visualized with FITC (FLAG-CHMP4b) or Cy3 (Core). Images were visualized using confocal laser scanning microscopy. The right panels exhibit the two-color overlay images (Merged). Colocalization is shown in yellow. (C) The Core or NS5A partially colocalizes with CHMP4b in HCV-JFH1-infected RSc cells. RSc cells transfected with 100 ng of pCHMP4b-GFP were infected with HCV-JFH1. Cells were fixed 60 hrs post-infection and were then examined by confocal laser scanning microscopy as shown in panel (A). (D) HCV Core binds to CHMP4b. 293FT cells transfected with 4 µg of pCHMP4b-GFP, pEGFP C3 (Clontech), pcDNA3-FLAG, pcDNA3-FLAG-Alix or pFLAG-CHMP4b and RSc cells 5 days after inoculation of HCV-JFH1 at an MOI of 4 were lysed. The mixtures of these lysates were immunoprecipitated with either anti-FLAG or Living Colors A.v. monoclonal antibody (anti-GFP antibody), followed by immunoblot analysis using anti-HCV Core, anti-HCV NS5A, anti-FLAG, and/or Living Colors A.v. monoclonal antibody.
Figure Legend Snippet: HCV Core interacts with CHMP4b. (A–C) HCV Core colocalizes with CHMP4b. 293FT cells cotransfected with 100 ng of pcDNA3/core (JFH1) and either 100 ng of pCHMP4b-GFP [39] (A) or pFLAG-CHMP4b [39] (B) were examined by confocal laser scanning microscopy. Cells were stained with anti-HCV Core and anti-FLAG polyclonal antibody and were then visualized with FITC (FLAG-CHMP4b) or Cy3 (Core). Images were visualized using confocal laser scanning microscopy. The right panels exhibit the two-color overlay images (Merged). Colocalization is shown in yellow. (C) The Core or NS5A partially colocalizes with CHMP4b in HCV-JFH1-infected RSc cells. RSc cells transfected with 100 ng of pCHMP4b-GFP were infected with HCV-JFH1. Cells were fixed 60 hrs post-infection and were then examined by confocal laser scanning microscopy as shown in panel (A). (D) HCV Core binds to CHMP4b. 293FT cells transfected with 4 µg of pCHMP4b-GFP, pEGFP C3 (Clontech), pcDNA3-FLAG, pcDNA3-FLAG-Alix or pFLAG-CHMP4b and RSc cells 5 days after inoculation of HCV-JFH1 at an MOI of 4 were lysed. The mixtures of these lysates were immunoprecipitated with either anti-FLAG or Living Colors A.v. monoclonal antibody (anti-GFP antibody), followed by immunoblot analysis using anti-HCV Core, anti-HCV NS5A, anti-FLAG, and/or Living Colors A.v. monoclonal antibody.

Techniques Used: Confocal Laser Scanning Microscopy, Staining, Infection, Transfection, Immunoprecipitation

28) Product Images from "Knocking down of heat-shock protein 27 directs differentiation of functional glutamatergic neurons from placenta-derived multipotent cells"

Article Title: Knocking down of heat-shock protein 27 directs differentiation of functional glutamatergic neurons from placenta-derived multipotent cells

Journal: Scientific Reports

doi: 10.1038/srep30314

Physiological effects of over-expression of HSP27 in PDMCs. The PDMCs were induced with IBMX for 6 h and then transfected with the pEGFP-C3 or pEGFP-C3/HSP27 plasmids, as indicated. ( A ) The cells were fixed and directly visualized using fluorescence microscopy at 1 day, 2 days and 3 days after transfection. The green cells show the expression of GFP. The red arrows indicate the neuron-like cells, and yellow arrows indicate the GFP-expressing cells. ( B ) Cells from each transfection were fixed and probed with Pan-Neuronal Marker (PNM) antibody for visualizing neuronal cells. The GFP/HSP27-transfected cells showed green signal alone, without co-localization with PNM-positive cells. ( C ) Without HSP27 overexpression, there was partial colocalization of the GFP and PNM signals. (Magnification = 200x). ( D ) The transfected cells in each condition were probed with PNM antibody, and PNM fluorescence signals were recorded and quantified. The cells showing PNM immunofluorescence were counted as positive cells. The proportion of differentiated neurons was quantified as the positive PNM immunofluorescence staining divided by the DAPI-positive cells. ( E ) Immunoblots of PDMCs with HSP27 overexpression. The upregulated HSP27 protein expression levels were confirmed in HSP27-overexpressing cells (right column) compared with controls. HSP27-overexpressing PDMCs (indicated by arrowheads) showed no cleaved forms of caspase-3 or Nanog. Additionally, the cleaved forms of SOX2 and c-myc, two other stem cell markers, were absent in both experimental conditions. GAPDH was used as a loading control for immunoblotting.
Figure Legend Snippet: Physiological effects of over-expression of HSP27 in PDMCs. The PDMCs were induced with IBMX for 6 h and then transfected with the pEGFP-C3 or pEGFP-C3/HSP27 plasmids, as indicated. ( A ) The cells were fixed and directly visualized using fluorescence microscopy at 1 day, 2 days and 3 days after transfection. The green cells show the expression of GFP. The red arrows indicate the neuron-like cells, and yellow arrows indicate the GFP-expressing cells. ( B ) Cells from each transfection were fixed and probed with Pan-Neuronal Marker (PNM) antibody for visualizing neuronal cells. The GFP/HSP27-transfected cells showed green signal alone, without co-localization with PNM-positive cells. ( C ) Without HSP27 overexpression, there was partial colocalization of the GFP and PNM signals. (Magnification = 200x). ( D ) The transfected cells in each condition were probed with PNM antibody, and PNM fluorescence signals were recorded and quantified. The cells showing PNM immunofluorescence were counted as positive cells. The proportion of differentiated neurons was quantified as the positive PNM immunofluorescence staining divided by the DAPI-positive cells. ( E ) Immunoblots of PDMCs with HSP27 overexpression. The upregulated HSP27 protein expression levels were confirmed in HSP27-overexpressing cells (right column) compared with controls. HSP27-overexpressing PDMCs (indicated by arrowheads) showed no cleaved forms of caspase-3 or Nanog. Additionally, the cleaved forms of SOX2 and c-myc, two other stem cell markers, were absent in both experimental conditions. GAPDH was used as a loading control for immunoblotting.

Techniques Used: Over Expression, Transfection, Fluorescence, Microscopy, Expressing, Marker, Immunofluorescence, Staining, Western Blot

29) Product Images from "Rabring7, a Novel Rab7 Target Protein with a RING Finger Motif"

Article Title: Rabring7, a Novel Rab7 Target Protein with a RING Finger Motif

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-08-0495

Subcellular distribution of Rabring7. (A) BHK cells were transfected with pcDNA/Rabring7 and pEGFP-C3 or pEGFP/Rab4wt, pEGFP/Rab7wt, pEGFP/Rab8wt. After 48 h, the cells were fixed and examined by GFP fluorescence for EGFP-Rabs and by immunofluorescence labeling for His-Rabring7. Top: fluorescence images of EGFP; middle: fluorescence images of His-Rabring7; bottom: merged images. Bar, 30 μm (B) BHK cells were transfected with pcDNA/Rabring7 and pEGFP/Rab7wt or pEGFP/Rab7Q67L, pEGFP/Rab7T22N, and pEGFP/Rab7N125I. After 48 h, the cells were fixed and examined by GFP fluorescence for EGFP-Rab7s and by immunofluorescence labeling for His-Rabring7. Top: fluorescence images of EGFP; middle: fluorescence images of His-Rabring7; bottom: merged images. Bar, 30 μm (C) BHK cells were transfected with pEGFP-C3 or pEGFP/Rab7wt. After 48 h, the cells were fixed and examined by GFP fluorescence for EGFP and EGFP-Rab7 and by immunofluorescence labeling for endogenous Rabring7. Top: fluorescence images of EGFP; middle: fluorescence images of endogenous Rabring7; bottom: merged images. Bar, 30 μm.
Figure Legend Snippet: Subcellular distribution of Rabring7. (A) BHK cells were transfected with pcDNA/Rabring7 and pEGFP-C3 or pEGFP/Rab4wt, pEGFP/Rab7wt, pEGFP/Rab8wt. After 48 h, the cells were fixed and examined by GFP fluorescence for EGFP-Rabs and by immunofluorescence labeling for His-Rabring7. Top: fluorescence images of EGFP; middle: fluorescence images of His-Rabring7; bottom: merged images. Bar, 30 μm (B) BHK cells were transfected with pcDNA/Rabring7 and pEGFP/Rab7wt or pEGFP/Rab7Q67L, pEGFP/Rab7T22N, and pEGFP/Rab7N125I. After 48 h, the cells were fixed and examined by GFP fluorescence for EGFP-Rab7s and by immunofluorescence labeling for His-Rabring7. Top: fluorescence images of EGFP; middle: fluorescence images of His-Rabring7; bottom: merged images. Bar, 30 μm (C) BHK cells were transfected with pEGFP-C3 or pEGFP/Rab7wt. After 48 h, the cells were fixed and examined by GFP fluorescence for EGFP and EGFP-Rab7 and by immunofluorescence labeling for endogenous Rabring7. Top: fluorescence images of EGFP; middle: fluorescence images of endogenous Rabring7; bottom: merged images. Bar, 30 μm.

Techniques Used: Transfection, Fluorescence, Immunofluorescence, Labeling

30) Product Images from "Identification of Nuclear Localization Signals in the ORF2 Protein of Porcine Circovirus Type 3"

Article Title: Identification of Nuclear Localization Signals in the ORF2 Protein of Porcine Circovirus Type 3

Journal: Viruses

doi: 10.3390/v11121086

The key amino acids of NLS in ORF2-1-3. ( A ) Mutants of ORF2-1-3 gene were inserted into pEGFP-C3, and the key amino acids predicted in ORF2-1-3 are listed by underline. ( B ) PK-15 cells were transfected with mutant plasmids, and the localization of fusion proteins was observed by confocal microscopy. Scale bars = 20 μm. ( C ) and ( D ) After 24 h, the nucleus and cytoplasm in transfected cells were extracted and the expressed proteins were determined by western blot. Nuclear/cytoplasmic distribution of the expressed proteins was further analyzed through densitometric quantification using Image-Pro software, data from three independent experiments are shown on the graph as the average ± standard error. One-way ANOVA; ** p
Figure Legend Snippet: The key amino acids of NLS in ORF2-1-3. ( A ) Mutants of ORF2-1-3 gene were inserted into pEGFP-C3, and the key amino acids predicted in ORF2-1-3 are listed by underline. ( B ) PK-15 cells were transfected with mutant plasmids, and the localization of fusion proteins was observed by confocal microscopy. Scale bars = 20 μm. ( C ) and ( D ) After 24 h, the nucleus and cytoplasm in transfected cells were extracted and the expressed proteins were determined by western blot. Nuclear/cytoplasmic distribution of the expressed proteins was further analyzed through densitometric quantification using Image-Pro software, data from three independent experiments are shown on the graph as the average ± standard error. One-way ANOVA; ** p

Techniques Used: Transfection, Mutagenesis, Confocal Microscopy, Western Blot, Software

The main nucleolar localization signal motifs in ORF2-1-2. ( A ) Mutants of ORF2-1-2 gene were inserted into pEGFP-C3 and predicted nucleolar localization signal motifs are underlined. ( B ) PK-15 cells were transfected with plasmids, after 24 h, the cells were fixed and the localization of fusion proteins was observed by confocal microscopy. Scale bars = 20 μm.
Figure Legend Snippet: The main nucleolar localization signal motifs in ORF2-1-2. ( A ) Mutants of ORF2-1-2 gene were inserted into pEGFP-C3 and predicted nucleolar localization signal motifs are underlined. ( B ) PK-15 cells were transfected with plasmids, after 24 h, the cells were fixed and the localization of fusion proteins was observed by confocal microscopy. Scale bars = 20 μm.

Techniques Used: Transfection, Confocal Microscopy

Related Articles

Transfection:

Article Title: Mycobacterial and HIV Infections Up-Regulated Human Zinc Finger Protein 134, a Novel Positive Regulator of HIV-1 LTR Activity and Viral Propagation
Article Snippet: .. For quantification of the LTR driven viral transcripts, second round of transfection was performed with pNL4-3 vector (40 ng/well) after 24 hours of transfection of pEGFP-C3 or ZNF-134-GFP-C3 vector. .. The transfection efficiencies of pNL4-3 were normalized with pRL-TK.

Article Title: Novel restriction factor RNA-associated early-stage anti-viral factor (REAF) inhibits human and simian immunodeficiency viruses
Article Snippet: .. Click here for file Additional file 4 Transfection efficiency of REAF-EGFP compared to pEGFP-C3. .. HeLa-CD4 cells transfected with REAF-EGFP or pEGFP-C3 empty vector.

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice
Article Snippet: .. The fraction of cells transfected with mACE2/pcDNA3.1 that was cotransfected with the ADAM17 expression plasmids was estimated by transfection of cells with 0.5 μg pACE2-Tom and cotransfection with 2 μg of either pAd17 or 2 μg pEGFP-C3 per well. .. The population of purely red fluorescent cells seen in the lower left panel of C is more than 99% shifted to cells coexpressing red and green fluorescence when pEGFP-C3 is cotransfected ( C, lower right panel).

Luciferase:

Article Title: Mycobacterial and HIV Infections Up-Regulated Human Zinc Finger Protein 134, a Novel Positive Regulator of HIV-1 LTR Activity and Viral Propagation
Article Snippet: .. For luciferase based assay, pLTR-luc (30 ng/well) and pRL-TK (5 ng/well) were co-transfected with pEGFP-C3 or ZNF-134-GFP-C3 (100 ng/well) or pcTAT (20 ng/well) separately or together. .. 48 hours post transfection, cells were harvested and analyzed for luciferase activity using Dual Glow Luciferase assay Kit as per the manufacturer's protocol (Promega, USA).

Expressing:

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice
Article Snippet: .. Other plasmids used were pcDNA3.1(−) (Invitrogen/Life Technologies) and pEGFP-C3 (CLONTECH) for cytomegalovirus (CMV) promoter-driven expression of enhanced GFP. .. Rat insulinoma 832/13 cells were transfected in six-well plates as previously described ( ) with varying amounts of expression plasmids.

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice
Article Snippet: .. The fraction of cells transfected with mACE2/pcDNA3.1 that was cotransfected with the ADAM17 expression plasmids was estimated by transfection of cells with 0.5 μg pACE2-Tom and cotransfection with 2 μg of either pAd17 or 2 μg pEGFP-C3 per well. .. The population of purely red fluorescent cells seen in the lower left panel of C is more than 99% shifted to cells coexpressing red and green fluorescence when pEGFP-C3 is cotransfected ( C, lower right panel).

Infection:

Article Title: Mycobacterial and HIV Infections Up-Regulated Human Zinc Finger Protein 134, a Novel Positive Regulator of HIV-1 LTR Activity and Viral Propagation
Article Snippet: .. Primer list is given in . pLTR-Luc (a gift from Dr. Debashish Mitra, NCCS, Pune, India); pNL4-3 : a full length replication and infection competent chimeric DNA (Gene Bank: AF324493) ; pcTat (a gift from Prof Anand K Kondapi), pRL-TK (Promega, USA) and pEGFP-C3 (Clonetech, USA) were used for the study. .. PBMC isolation and infections with NL4-3 and Mycobacterium bovis BCG All experiments were performed in the facilities approved for Mycobacterial and HIV cultures by University of Hyderabad Institutional Biosafety Committee under Department of Biotechnology, Govt. of India (UH/SLS/IBSC/Review/facilities F-60 & F-70).

Generated:

Article Title: Intramembrane Proteolysis and Endoplasmic Reticulum Retention of Hepatitis C Virus Core Protein
Article Snippet: .. The genes encoding core proteins with the region between amino acids 128 and 151 deleted and replacement of Leu139 , Val140 , and Leu144 with Ala were generated by the method of splicing by overlap extension ( , , ) and introduced into pEGFP-C3; these constructs are designated EGFP-Core Δ128-151 and EGFP-Core LVL/3A, respectively (Fig. ). ..

Overlap Extension Polymerase Chain Reaction:

Article Title: Intramembrane Proteolysis and Endoplasmic Reticulum Retention of Hepatitis C Virus Core Protein
Article Snippet: .. The genes encoding core proteins with the region between amino acids 128 and 151 deleted and replacement of Leu139 , Val140 , and Leu144 with Ala were generated by the method of splicing by overlap extension ( , , ) and introduced into pEGFP-C3; these constructs are designated EGFP-Core Δ128-151 and EGFP-Core LVL/3A, respectively (Fig. ). ..

Construct:

Article Title: Intramembrane Proteolysis and Endoplasmic Reticulum Retention of Hepatitis C Virus Core Protein
Article Snippet: .. The genes encoding core proteins with the region between amino acids 128 and 151 deleted and replacement of Leu139 , Val140 , and Leu144 with Ala were generated by the method of splicing by overlap extension ( , , ) and introduced into pEGFP-C3; these constructs are designated EGFP-Core Δ128-151 and EGFP-Core LVL/3A, respectively (Fig. ). ..

Cotransfection:

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice
Article Snippet: .. The fraction of cells transfected with mACE2/pcDNA3.1 that was cotransfected with the ADAM17 expression plasmids was estimated by transfection of cells with 0.5 μg pACE2-Tom and cotransfection with 2 μg of either pAd17 or 2 μg pEGFP-C3 per well. .. The population of purely red fluorescent cells seen in the lower left panel of C is more than 99% shifted to cells coexpressing red and green fluorescence when pEGFP-C3 is cotransfected ( C, lower right panel).

Plasmid Preparation:

Article Title: Mycobacterial and HIV Infections Up-Regulated Human Zinc Finger Protein 134, a Novel Positive Regulator of HIV-1 LTR Activity and Viral Propagation
Article Snippet: .. For quantification of the LTR driven viral transcripts, second round of transfection was performed with pNL4-3 vector (40 ng/well) after 24 hours of transfection of pEGFP-C3 or ZNF-134-GFP-C3 vector. .. The transfection efficiencies of pNL4-3 were normalized with pRL-TK.

Article Title: Novel restriction factor RNA-associated early-stage anti-viral factor (REAF) inhibits human and simian immunodeficiency viruses
Article Snippet: .. The vector used was pEGFP-C3 (Clontech). ..

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • egfp  (TaKaRa)
    92
    TaKaRa egfp
    Expression of the intronic shINX1 plasmid selectively reduces Hve–inx1 mRNA levels and uncouples the AP neuron. A , B , Double labeling of embryonic AP neurons 48 h after expression of the <t>EGFP–shINX1</t> reporter vector ( A1–A3 ; asterisks indicate AP neurons) and in situ hybridization (ISH; B1–B3 ), revealing the loss of INX1 mRNA staining (red arrowheads). C , D , Control expression of an shRNA targeting INX19 ( <t>C1–C3</t> ; asterisks) fails to diminish INX1 mRNA labeling intensity ( D1–D3 ; red arrowheads). E , Quantification of INX1 mRNA staining intensity after shINX1 and INX19 expression. Values represent gray levels above background, corresponding to the location of the APs (transformed neurons; black bars) or nontransformed neurons adjacent to the APs (non-transformed neurons; gray bars). Data are ±SD (** p
    Egfp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egfp/product/TaKaRa
    Average 92 stars, based on 83 article reviews
    Price from $9.99 to $1999.99
    egfp - by Bioz Stars, 2020-05
    92/100 stars
      Buy from Supplier

    91
    TaKaRa pegfp c3
    hZNF-134 over expression enhanced the transcriptional potential of LTR and increased viral titers. (A) Luciferase activity was measured in the presence or absence of Tat following transfection with <t>pEGFP-C3</t> or ZNF-134-GFP-C3 vector into HEK293T cells along with the reporter construct, pLTR-luc. Luciferase activity was measured in terms of relative luminescence units (RLU) from HEK293T cell lysates after 48 hours and plotted as fold increase in the luciferase activity. Cells over-expressing hZNF-134-GFP showed increased luciferase activity when compared to GFP alone. (B) pNL4-3 was transfected in HEK293T cells with either GFP or hZNF-134-GFP. After 48 hours, the viral transcription was measured by qRT-PCR. The plot shows that viral transcripts increased in the presence of over-expressed hZNF-134. (C) HEK293T cells over-expressing either GFP or hZNF-134-GFP were transfected with pNL4-3 vector and after 48 hours scored for viral p24 by ELISA. Inset shows the Western blot confirming the over-expression of either GFP or hZNF-134-GFP in HEK293T cells. For loading control, expression of GAPDH was detected by Western blots in both the cases. Over-expression of hZNF-134 increased viral titers. All the transfection efficiencies were normalized to pRL-TK transfections. All experiments were done more than three times and error bars represents mean ± SD. Student's t -test was performed and *p
    Pegfp C3, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp c3/product/TaKaRa
    Average 91 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    pegfp c3 - by Bioz Stars, 2020-05
    91/100 stars
      Buy from Supplier

    86
    TaKaRa expression plasmid pegfp c3
    Cells over-expressing non-catalytic GFP-GAP273 migrate more slowly in an in vitro wound healing assay. Monolayers of HEK293 cells transiently transfected with GFP-GAP273, GFP-GalT, or <t>pEGFP-C3</t> empty vector were scratched and the cell were allowed to spread into the scratched area. The degree of cell migration into scratched area was recorded at the indicated times. A. Images of cell migration over time. B. The average widths of the scratched area measured at various points are graphed. (n = 5, Error bars = S.D.)
    Expression Plasmid Pegfp C3, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/expression plasmid pegfp c3/product/TaKaRa
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    expression plasmid pegfp c3 - by Bioz Stars, 2020-05
    86/100 stars
      Buy from Supplier

    Image Search Results


    Expression of the intronic shINX1 plasmid selectively reduces Hve–inx1 mRNA levels and uncouples the AP neuron. A , B , Double labeling of embryonic AP neurons 48 h after expression of the EGFP–shINX1 reporter vector ( A1–A3 ; asterisks indicate AP neurons) and in situ hybridization (ISH; B1–B3 ), revealing the loss of INX1 mRNA staining (red arrowheads). C , D , Control expression of an shRNA targeting INX19 ( C1–C3 ; asterisks) fails to diminish INX1 mRNA labeling intensity ( D1–D3 ; red arrowheads). E , Quantification of INX1 mRNA staining intensity after shINX1 and INX19 expression. Values represent gray levels above background, corresponding to the location of the APs (transformed neurons; black bars) or nontransformed neurons adjacent to the APs (non-transformed neurons; gray bars). Data are ±SD (** p

    Journal: The Journal of Neuroscience

    Article Title: Gap Junction-Dependent Homolog Avoidance in the Developing CNS

    doi: 10.1523/JNEUROSCI.2387-13.2013

    Figure Lengend Snippet: Expression of the intronic shINX1 plasmid selectively reduces Hve–inx1 mRNA levels and uncouples the AP neuron. A , B , Double labeling of embryonic AP neurons 48 h after expression of the EGFP–shINX1 reporter vector ( A1–A3 ; asterisks indicate AP neurons) and in situ hybridization (ISH; B1–B3 ), revealing the loss of INX1 mRNA staining (red arrowheads). C , D , Control expression of an shRNA targeting INX19 ( C1–C3 ; asterisks) fails to diminish INX1 mRNA labeling intensity ( D1–D3 ; red arrowheads). E , Quantification of INX1 mRNA staining intensity after shINX1 and INX19 expression. Values represent gray levels above background, corresponding to the location of the APs (transformed neurons; black bars) or nontransformed neurons adjacent to the APs (non-transformed neurons; gray bars). Data are ±SD (** p

    Article Snippet: A short-hairpin RNA (shRNA) expression cassette was designed with a leech actin intron ( Hm Act1; GenBank accession number , base pairs 1801–1956) inserted downstream of the ORF of EGFP (pEGFP–C3; Clontech) between the BglII and BamHI sites of the multiple cloning region.

    Techniques: Expressing, Plasmid Preparation, Labeling, In Situ Hybridization, Staining, shRNA, Transformation Assay

    hZNF-134 over expression enhanced the transcriptional potential of LTR and increased viral titers. (A) Luciferase activity was measured in the presence or absence of Tat following transfection with pEGFP-C3 or ZNF-134-GFP-C3 vector into HEK293T cells along with the reporter construct, pLTR-luc. Luciferase activity was measured in terms of relative luminescence units (RLU) from HEK293T cell lysates after 48 hours and plotted as fold increase in the luciferase activity. Cells over-expressing hZNF-134-GFP showed increased luciferase activity when compared to GFP alone. (B) pNL4-3 was transfected in HEK293T cells with either GFP or hZNF-134-GFP. After 48 hours, the viral transcription was measured by qRT-PCR. The plot shows that viral transcripts increased in the presence of over-expressed hZNF-134. (C) HEK293T cells over-expressing either GFP or hZNF-134-GFP were transfected with pNL4-3 vector and after 48 hours scored for viral p24 by ELISA. Inset shows the Western blot confirming the over-expression of either GFP or hZNF-134-GFP in HEK293T cells. For loading control, expression of GAPDH was detected by Western blots in both the cases. Over-expression of hZNF-134 increased viral titers. All the transfection efficiencies were normalized to pRL-TK transfections. All experiments were done more than three times and error bars represents mean ± SD. Student's t -test was performed and *p

    Journal: PLoS ONE

    Article Title: Mycobacterial and HIV Infections Up-Regulated Human Zinc Finger Protein 134, a Novel Positive Regulator of HIV-1 LTR Activity and Viral Propagation

    doi: 10.1371/journal.pone.0104908

    Figure Lengend Snippet: hZNF-134 over expression enhanced the transcriptional potential of LTR and increased viral titers. (A) Luciferase activity was measured in the presence or absence of Tat following transfection with pEGFP-C3 or ZNF-134-GFP-C3 vector into HEK293T cells along with the reporter construct, pLTR-luc. Luciferase activity was measured in terms of relative luminescence units (RLU) from HEK293T cell lysates after 48 hours and plotted as fold increase in the luciferase activity. Cells over-expressing hZNF-134-GFP showed increased luciferase activity when compared to GFP alone. (B) pNL4-3 was transfected in HEK293T cells with either GFP or hZNF-134-GFP. After 48 hours, the viral transcription was measured by qRT-PCR. The plot shows that viral transcripts increased in the presence of over-expressed hZNF-134. (C) HEK293T cells over-expressing either GFP or hZNF-134-GFP were transfected with pNL4-3 vector and after 48 hours scored for viral p24 by ELISA. Inset shows the Western blot confirming the over-expression of either GFP or hZNF-134-GFP in HEK293T cells. For loading control, expression of GAPDH was detected by Western blots in both the cases. Over-expression of hZNF-134 increased viral titers. All the transfection efficiencies were normalized to pRL-TK transfections. All experiments were done more than three times and error bars represents mean ± SD. Student's t -test was performed and *p

    Article Snippet: Primer list is given in . pLTR-Luc (a gift from Dr. Debashish Mitra, NCCS, Pune, India); pNL4-3 : a full length replication and infection competent chimeric DNA (Gene Bank: AF324493) ; pcTat (a gift from Prof Anand K Kondapi), pRL-TK (Promega, USA) and pEGFP-C3 (Clonetech, USA) were used for the study.

    Techniques: Over Expression, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Construct, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot

    hZNF-134-GFP is localized in the nucleus of HEK293T and Astrocytoma 1321N1 cells. HEK293T and 1321N1 cells were transfected with expression plasmids pEGFP-C3 or ZNF-134-GFP-C3. The cells were fixed after 48 hours and visualized under confocal microscopy. Panels show Trans, DAPI, GFP, and Merged images. hZNF-134-GFP protein was localized in the nucleus of HEK293T and 1321N1 cells whereas GFP control showed both nuclear and cytoplasmic localization. Localization was confirmed by 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Mycobacterial and HIV Infections Up-Regulated Human Zinc Finger Protein 134, a Novel Positive Regulator of HIV-1 LTR Activity and Viral Propagation

    doi: 10.1371/journal.pone.0104908

    Figure Lengend Snippet: hZNF-134-GFP is localized in the nucleus of HEK293T and Astrocytoma 1321N1 cells. HEK293T and 1321N1 cells were transfected with expression plasmids pEGFP-C3 or ZNF-134-GFP-C3. The cells were fixed after 48 hours and visualized under confocal microscopy. Panels show Trans, DAPI, GFP, and Merged images. hZNF-134-GFP protein was localized in the nucleus of HEK293T and 1321N1 cells whereas GFP control showed both nuclear and cytoplasmic localization. Localization was confirmed by 3 independent experiments.

    Article Snippet: Primer list is given in . pLTR-Luc (a gift from Dr. Debashish Mitra, NCCS, Pune, India); pNL4-3 : a full length replication and infection competent chimeric DNA (Gene Bank: AF324493) ; pcTat (a gift from Prof Anand K Kondapi), pRL-TK (Promega, USA) and pEGFP-C3 (Clonetech, USA) were used for the study.

    Techniques: Transfection, Expressing, Confocal Microscopy

    Induction of PC12 differentiation by Raf proteins. PC12 cells were cotransfected with the indicated pCDNA3-derived Raf constructs and the pEGFP-C3 reporter construct. The indicated percentages represent the ratios between green fluorescent protein (GFP)-positive cells undergoing neurite outgrowth and the total number of GFP-positive transfected cells. WT, wild type.

    Journal: Molecular and Cellular Biology

    Article Title: A Raf-1 Mutant That Dissociates MEK/Extracellular Signal-Regulated Kinase Activation from Malignant Transformation and Differentiation but Not Proliferation

    doi: 10.1128/MCB.23.6.1983-1993.2003

    Figure Lengend Snippet: Induction of PC12 differentiation by Raf proteins. PC12 cells were cotransfected with the indicated pCDNA3-derived Raf constructs and the pEGFP-C3 reporter construct. The indicated percentages represent the ratios between green fluorescent protein (GFP)-positive cells undergoing neurite outgrowth and the total number of GFP-positive transfected cells. WT, wild type.

    Article Snippet: Cells were cotransfected with 3.0 μg of pcDNA3-derived constructs and 0.2 μg of pEGFP-C3 reporter construct, encoding enhanced green fluorescent protein (Clontech) by using Lipofectamin 2000 reagent as recommended by the manufacturer (Invitrogen).

    Techniques: Derivative Assay, Construct, Transfection

    Cells over-expressing non-catalytic GFP-GAP273 migrate more slowly in an in vitro wound healing assay. Monolayers of HEK293 cells transiently transfected with GFP-GAP273, GFP-GalT, or pEGFP-C3 empty vector were scratched and the cell were allowed to spread into the scratched area. The degree of cell migration into scratched area was recorded at the indicated times. A. Images of cell migration over time. B. The average widths of the scratched area measured at various points are graphed. (n = 5, Error bars = S.D.)

    Journal: PLoS ONE

    Article Title: The Non-Catalytic Carboxyl-Terminal Domain of ARFGAP1 Regulates Actin Cytoskeleton Reorganization by Antagonizing the Activation of Rac1

    doi: 10.1371/journal.pone.0018458

    Figure Lengend Snippet: Cells over-expressing non-catalytic GFP-GAP273 migrate more slowly in an in vitro wound healing assay. Monolayers of HEK293 cells transiently transfected with GFP-GAP273, GFP-GalT, or pEGFP-C3 empty vector were scratched and the cell were allowed to spread into the scratched area. The degree of cell migration into scratched area was recorded at the indicated times. A. Images of cell migration over time. B. The average widths of the scratched area measured at various points are graphed. (n = 5, Error bars = S.D.)

    Article Snippet: Green fluorescent fusion proteins were made by inserting various fragments of ARFGAP1 cDNA into the expression plasmid pEGFP-C3 (Clontech).

    Techniques: Expressing, In Vitro, Wound Healing Assay, Transfection, Plasmid Preparation, Migration